Effect of prosthetic restoration on masticatory function in patients with shortened dental arches: a multicentre study.

The aim of this multicentre study was to investigate the effect of prosthetic restoration for missing posterior teeth on mastication in patients with shortened dental arches (SDAs). Partially dentate patients who had an intact teeth in anterior region and missed distal molar(s) (2-12 missing occlusal units) classified as Kennedy Class I or Class II were recruited from seven university-based dental hospitals in Japan. Of the 125 subjects who underwent baseline (pre-treatment) and follow-up/post-treatment evaluation, 53 chose no replacement of missing teeth and 72 chose treatment with removable partial dentures (n = 53) or implant-supported fixed partial dentures (n = 19). Objective masticatory performance (MP) was evaluated using a gummy jelly test. Perception of chewing ability (CA) was rated using a food intake questionnaire. In the no-treatment group, mean MP and CA scores at baseline were similar to those at follow-up evaluation (P > 0·05). In the treatment group, mean MP after treatment was significantly greater than the pre-treatment mean MP (P < 0·05). However, the mean perceived CA in the treatment groups was similar at pre- and post-treatment (P > 0·05). In a subgroup analysis of subjects in the treatment group, subjects with lower pre-treatment CA showed a significant CA increase after treatment (P = 0·004), but those with higher pre-treatment CA showed a significant decrease in CA (P = 0·001). These results suggest that prosthetic restoration for SDAs may benefit objective masticatory performance in patients needing replacement of missing posterior teeth, but the benefit in subjective chewing ability seems to be limited in subjects with perceived impairment in chewing ability before treatment.

Strontium hydroxyapatite in situ gel-forming system - a new approach for minimally invasive bone augmentation.

OBJECTIVES: To achieve ideal functional and aesthetic requirements, ridge augmentation is often required before dental implant placement. Bone augmentation (especially vertical), which normally consists of complex and invasive surgeries, still remains challenge. This study seeks to investigate the feasibility of an injectable in situ gel-forming system containing strontium hydroxyapatite (SrHA) and alginate for minimally invasive bone augmentation in a rat calvarial model. MATERIAL AND METHODS: SrHA-alginate solution was prepared by mixing SrHA powder with alginate solution (20 mg/mL) to the final concentration of 0.5% (w/v). Each animal received a 200-µL single subperiosteal injection of either SrHA-alginate solution or alginate solution. The new bone formation was assessed at 0, 4, and 8 weeks histologically and radiologically. RESULTS: The SrHA-alginate solution materials could form solid gel once injected. As such, no sutures were required to close the injection site. Significantly greater amount of new bone formation was observed in the SrHA-alginate group compared with the alginate group both by micro-CT and by histological section. The newly formed bone in the SrHA-alginate group originated both from the underlying original bone and from the elevated periosteum. A 2.3-fold increase of the vertical bone height was observed in the SrHA-alginate group compared with 1.3-fold increase in the alginate group. CONCLUSIONS: Rat calvarial bone augmentation was achieved by a single subperiosteal injection of SrHA-alginate solution without any administration of stem cells or growth factors. The in situ gel-forming material may hold potential therapeutic benefits for local bone augmentation in a minimally invasive manner.

Novel bioresorbable strontium hydroxyapatite membrane for guided bone regeneration.

OBJECTIVES: Membrane materials have been widely used for guided bone regeneration (GBR). However, due to bio-functional limitation of the current membranes, the ideal resorbable membrane that can stimulate bone regeneration has yet to be developed. This study seeks to investigate the effects of a strontium hydroxyapatite (SrHA)-containing membrane for GBR. MATERIAL AND METHODS: Strontium hydroxyapatite powder was synthesized and mixed with gelatin solution to the final concentration of 10 mg/ml (Sr10) and 20 mg/ml (Sr20). Approximately 100-µm-thick membranes were fabricated, and the mechanical properties and strontium ion release pattern were analyzed. Rat bone marrow stromal cell (BMSC) responses were investigated in vitro. Bilaterial rat calvarial defects were used in vivo to compare the SrHA membranes against commercially available collagen membranes and evaluated radiologically and histologically. RESULTS: Strontium hydroxyapatite membranes exhibited higher elasticity and strength than the collagen membrane, and slow strontium ion release was also confirmed. No BMSC cytotoxicity was found on the SrHA membranes, and the alkaline phosphatase positively stained area was significantly greater than the collagen membrane at earlier time point. At 4 weeks, both micro-CT and histological analyses revealed that the Sr20 group yielded significantly greater bone formation. CONCLUSIONS: The SrHA-containing membrane developed in this study was found to be a biocompatible material that can stimulate BMSC differentiation as well as bone regeneration and maturation in rat calvarial defects at early time point compared with collagen membrane. The best result was observed in Sr20 group, which can be potentially effective for GBR.

Effect of prosthetic restoration on oral health-related quality of life in patients with shortened dental arches: a multicentre study.

The aim of this multicentre prospective study was to investigate the effect of prosthetic restoration for missing posterior teeth in patients with shortened dental arches (SDAs). SDA patients with 2-12 missing occlusal units (a pair of occluding premolars corresponds to one unit, and a pair of occluding molars corresponds to two units) were consecutively recruited from seven university-based dental hospitals in Japan. Patients chose no replacement of missing teeth or prosthetic treatment with removable partial dentures (RPDs) or implant-supported fixed partial dentures (IFPDs). Oral health-related quality of life (OHRQoL) was measured using the oral health impact profile (Japanese version - OHIP-J) at baseline and follow-up/post-treatment evaluation. Of the 169 subjects who completed baseline evaluation, 125 subjects (mean age; 63.0 years) received follow-up/post-treatment evaluation. No-treatment was chosen by 42% (53/125) of the subjects, and 58% (72/125) chose treatment with a RPD (n = 53) or an IFPD (n = 19). In the no-treatment (NT) group, the mean OHIP summary score at baseline was similar to that at follow-up evaluation (P = 0.69). In the treatment (TRT) group, the mean OHIP summary score decreased significantly after the RPD treatment (P = 0.002), and it tended to decrease, though not statistically significant (P = 0.18), after the IFPD treatment. The restoration of one occlusal unit was associated with a 1.2-point decrease in OHIP summary score (P = 0.034). These results suggest that the replacement of missing posterior teeth with RPDs or IFPDs improved OHRQoL. Prosthetic restoration for SDAs may benefit OHRQoL in patients needing replacement of missing posterior teeth.

The effect of glutathione on 2-hydroxyethylmethacrylate cytotoxicity and on resin-dentine bond strength.

AIM: To evaluate the influence of reduced glutathione (GSH) application on 2-hydroxyethylmethacrylate (HEMA) cytotoxicity on rat pulpal cells and evaluate the effect of etched-dentine treatment with GSH on the immediate microtensile bond strength (µTBS) of etch-and-rinse adhesive. METHODOLOGY: The cytotoxicity of 10 mmol L(-1) HEMA, 10 mmol L(-1) HEMA + 1 mmol L(-1) GSH, 10 mmol L(-1) HEMA + 5 mmol L(-1) GSH and 10 mmol L(-1) HEMA + 10 mmol L(-1) GSH was compared (6 h and 24 h). Cells viability was measured by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, followed by morphological observation of cells. Etched-dentine surfaces were rinsed and treated with one of the following solutions: 2% GSH, 5% GSH or 10% GSH, bonded with Adper Single Bond Plus (3M, ESPE, St. Paul, MN, USA) and restored with resin composite. The control group received no GSH treatment. After 1 day of water-storage at 37 °C, the specimens were subjected to µTBS testing. Cytotoxicity and µTBS data were analysed by one-way anova and Tukey post hoc tests (P < 0.05). RESULTS: There were significant differences between the groups. HEMA elicited a remarkable toxic effect. 10 mmol L(-1) GSH prevented HEMA-induced damage at both exposure times. Whilst 5 mmol L(-1) GSH lost its protective effect at 24-h exposure time and 1 mmol L(-1) GSH showed no protective effect at both exposure times, GSH had no significant effect on the immediate µTBS; however, 5% GSH had higher bond strength value when compared to 10% GSH (P = 0.003). CONCLUSION: Controlled concentrations of GSH had a protective effect against HEMA cytotoxicity. GSH had neither positive nor negative influence on µTBS.

Restricted expression of chromatin remodeling associated factor Chd3 during tooth root development.

BACKGROUND AND OBJECTIVE: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. MATERIAL AND METHODS: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. RESULTS: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig's epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. CONCLUSION: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.

Enhanced osteoblast and osteoclast responses to a thin film sputtered hydroxyapatite coating.

A sputtering technique followed by a low temperature hydrothermal treatment has been demonstrated to produce a dense-and-bioactive hydroxyapatite thin film coating. The purpose of the present study was to investigate osteoblast and osteoclast responses to the hydroxyapatite coated plates and titanium plates with similar roughness. Rat bone marrow stromal cells were cultured on these plates to induce osteoblasts. The cells showed a significantly enhanced proliferation on the hydroxyapatite surface, accompanied by increase of osteoblastic phenotypes. The co-cultured osteoclasts exhibited the significantly different cell number and morphology between the hydroxyapatite and the titanium surfaces. A series of osteoclast marker genes were more stimulated on the hydroxyapatite and thirty two percent of the hydroxyapatite surface area could be resorbed by osteoclasts. The thin film sputtered hydroxyapatite could provide a favorable surface for both osteoblast and osteoclast formation and their function, indicating its good osteoconductivity and biodegradability.

Factors related to prosthetic restoration in patients with shortened dental arches: a multicentre study.

The aim of this study was to identify the factors related to prosthetic restoration in patients with shortened dental arches (SDA). SDA patients with 2-12 missing occlusal units were consecutively enrolled from seven university-based dental hospitals in Japan. Of the 145 subjects (mean age; 63·4 years), 41% chose no treatment and 59% sought to replace their edentulous spaces with removable partial dentures or implant-supported fixed partial dentures. Restoration decisions were related to tooth loss patterns. Only 3% of subjects missing just second molar(s) sought to receive prosthetic treatment, while the percentage increased to 58% in subjects who were missing first and second molars and 93% in subjects missing premolar(s). Logistic regression analyses found that young age, increased number of missing occlusal units, asymmetric arch and presence of chewing complaint were significant predictors for prosthetic restoration (P<0·05). Increased number of missing occlusal units and asymmetric arch were significant predictors for the presence of chewing complaint (P<0·05). These results suggest that perceived impairment of chewing ability owing to missing occlusal units is a critical factor for prosthetic restoration in SDA patients.

Selective drug delivery system to bone: small peptide (Asp)6 conjugation.

Targeting a drug on hydroxyapatite (HA) could be a promising way for selective drug delivery to bone, because HA, an inorganic component in hard tissues (bone and teeth), does not exist in soft tissues. Several bone noncollagenous proteins, which bind to HA, have repeating sequences of acidic amino acids in their structures as possible HA-binding sites. Thus, we think that a small peptide of repetitive acidic amino acid could work as a carrier for selective drug delivery to the bone. To test this hypothesis, we conjugated (Asp)6 to fluorescein isothiocyanate (FITC), evaluated its affinity to HA in vitro, and examined its tissue distribution after injection into rats. Although fluorescein itself did not bind to HA, (Asp)6-FITC bound to HA as well as calceine and tetracycline. Twenty-four hours after intravenous injection of (Asp)6-FITC to rats, animals were killed, and ground sections of hard tissues and cryosections of soft tissues were made. Under a confocal laser scanning microscope, clear labeling lines were observed in bones and teeth, whereas no labeling was detected in soft tissues. In the rats administered with fluorescein alone, the fluorescent labeling was detected in neither hard nor soft tissues. Fluorescent analysis of blood, urine, and bones after (Asp)6-FITC administration revealed that biological half-life of FITC in blood was short (60 minutes) and that within 24 h, 95% of the administered FITC was excreted as urine whereas 2% of the FITC accumulated in bones. After subcutaneous administration of (Asp)6-FITC to mice, fluorescent intensity remaining in the femurs was measured periodically. In these mice the biological half-life of FITC in the femur was 14 days. Present results indicate that (Asp)6 is effective as a carrier for selective drug delivery to bone.

Evaluation of implant materials (hydroxyapatite, glass-ceramics, titanium) in rat bone marrow stromal cell culture.

Bone marrow stromal cells of femora from young adult rats produce bone-like mineralized tissue in culture. We evaluated three implant materials (hydroxyapatite (HA), glass-ceramics (GC) and commercial pure titanium (Ti)), as to their ability to provide an environment for marrow cells to differentiate into osteoblasts and function as suitable for mineralized tissue formation. We did this by measuring the DNA content, alkaline phosphatase (ALP) activity and calcium (Ca) content in culture, and the expression of osteopontin and bone sialoprotein by means of analysis of gene expression using Northern hybridization. DNA measurement showed no difference between each material, but ALP activity and Ca content in the culture on HA and GC were higher than on Ti and the control. An analysis of the gene expression revealed the highest expression of osteopontin and bone sialoprotein in the culture on HA. Mineralized nodule formation (both in area and number) was the most predominant on HA, followed by that on GC. These results showed that HA and GC provided a favourable situation for marrow cells to differentiate osteoblasts, resulting in a large amount of mineralized tissue formation on these surfaces. Present in vitro results could explain the rapid bone bonding of HA and GC in vivo.

Application of the MTT colorimetric assay to measure cytotoxic effects of phenolic compounds on established rat dental pulp cells.

The cytotoxicities of various phenolic compounds were examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay and established rat dental pulp cells (RPC-C2A). The ID50 (50% inhibition concentration) of the phenolic compounds ranged from 0.1 to 10 mmol/L, and the cytotoxicities of the phenolic compounds were related to their phenolic coefficients. The therapeutic index of each phenolic compound was calculated from the ID50 results and from the 50% inhibition concentration for prostaglandin synthesis that has been reported previously. Eugenol, guaiacol, and creosol had therapeutic indices much higher than those of other phenolic compounds examined. This experimental system could be used as a simple preliminary assay to test for toxicity of dental drugs and materials.

cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues.

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

A mineralizing rat dental pulp cell subline expressing collagen type I and dentin sialoprotein-phosphophoryn transcripts.

A mineralizing rat dental pulp cell subclone, termed M2H4, was selected from single-cell cloning of the rat dental pulp cell line RPC-C2A by screening confluent single-cell cultures for their ability to undergo mineralization. To induce mineralization, confluent single-cell cultures were treated for 8 d with ascorbic acid followed by the addition of inorganic phosphate to a final concentration of 4 mM for an additional 3 d. Confluent M2H4 subclones were shown by immunofluorescence and electron microscopy to form collagen type I fibrils. Furthermore, using reverse transcriptase-polymerase chain reaction, this subclone was found to be capable of expressing dentin sialoprotein-phosphophoryn (DSP-PP) transcripts, an odontoblast-specific marker. Thus, this newly identified mineralizing rat M2H4 subclone possesses odontoblast-like characteristics and can serve as an in vitro model for examining the role of DSP and PP in the formation of mineralized dentin.

Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture.

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

The effects of cytoskeletal inhibitors on the collagen gel contraction by dog periodontal ligament fibroblasts in vitro.

Periodontal ligament fibroblasts (PLF) were incorporated into small collagen gel discs (100 microliters, dia 10.0 mm), cultured in media containing various concentrations of cytochalasin D or colchicine and the diameters of the discs then measured. Cytochalasin D (3.1-800 nM) inhibited contraction of the gel discs in a dose-dependent manner and 800 nM of cytochalasin D completely inhibited contraction. This complete inhibition was still observed when cell density in the gel was increased. Although colchicine at a concentration of 0.98-15.7 nM also inhibited the contraction in a dose-dependent manner, contraction at 50% of the control level was still observed in the presence of a high concentration (0.25 mM, 100 micrograms/ml). These results strongly indicate that actin filaments are more intimately involved in PLF-mediated collagen gel contraction than microtubules. Although it is suggested that PLF are involved in tooth eruption, earlier studies have shown that microtubule inhibitors inhibit eruption of rat incisors whereas the microfilament inhibitor, cytochalasin B, has no effect. Thus, the PLF theory alone seems insufficient to explain the mechanism of tooth eruption.

Establishment and characterization of a clonal cell line (RPC-C2A) from dental pulp of the rat incisor.

Maxillary incisor pulp cells from male Wistar rats (7 weeks old) were cultured. After the 52nd subculture, cloning was performed twice and a clonal cell line (RPC-C2A) with high alkaline phosphatase (ALP) activity was established. The population doubling time of RPC-C2A cells was 13.7 h, and the mode of the chromosome number was 42. The heat stability of ALP, the effect of the ALP inhibitors, and the ALP polyacrylamide gel electrophoresis pattern in RPC-C2A cells was the same as for the ALP of the isolated dental pulp.

Characterization of a system of mineralized-tissue formation by rat dental pulp cells in culture.

Pulp tissue was obtained from maxillary incisors of young adult male Wistar rats, minced and digested with 0.5% trypsin and 0.02% EGTA at 37 degrees C for 30 min. Dissociated cells were cultured with or without 10 nM dexamethasone using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. Confluent cells were subcultured at 7 days and the medium further supplemented with beta-glycerophosphate (beta-GP). Dexamethasone in primary culture and/or secondary culture enhanced the formation of mineralized tissue while > 5 mM beta-GP was necessary for mineralization to occur. Biochemical analysis of the radiolabelled medium revealed that these cells produced type I, type I trimer and type III collagens. Analysis of [32PO4]-labelled medium, using DEAE-Sephacel ion-exchange chromatography and sodium dodecylsulphate-polyacrylamide gel electrophoresis, showed that these cells produced phosphophoryn-like protein. These results indicate that some of the rat dental pulp cells in culture express an odontoblast-like phenotype.

Measurements of the isometric contractile forces generated by dog periodontal ligament fibroblasts in vitro.

One hypothesis for the mechanism of tooth eruption is that the periodontal ligament fibroblasts generate the eruptive force. To assess the force generated, these fibroblasts were obtained by explant culture of ligament from mandibular premolars of a dog and were cultured in collagen gel matrices. The forces generated by them under isometric conditions were continuously measured for 120 h with a strain gauge. At the same time the number of cells in the gel was counted and the force measured was calculated as the force generated by 10(4) cells. Shortly after the start of culture, the force per 10(4) cells increased rapidly; it reached 5.2 X 10(-4) N at 8 h, and then remained at the same level for about 48 h. Our findings suggest that fibroblasts of the periodontal ligament may generate sufficient force for tooth eruption.

Effects of mechanical stress on the mRNA expression of S100A4 and cytoskeletal components by periodontal ligament cells.

The periodontal ligament (PDL) functions under constant mechanical stress, and PDL cells obviously control PDL functions under such conditions. We have previously found that the mRNA expression of the Ca2+-binding protein S100A4 and beta-actin is higher in the PDL from erupted teeth than in the PDL from teeth under eruption. This suggested a role for S100A4 in the response of PDL cells to mechanical stress, possibly by coupling Ca2+ and the cytoskeletal system. In the present study, we investigated the direct effects of cyclical stretching on the mRNA expression of S100A4 and two cytoskeletal components (beta-actin and alpha-tubulin) by PDL cells. In Northern blotting analysis, the expression of S100A4, beta-actin, and alpha-tubulin mRNAs was higher in the PDL from fully erupted and functional bovine teeth than in partially erupted ones. Similarly, when bovine PDL cells were mechanically stimulated by means of the Flexercell Strain Unit, the expression of S100A4, beta-actin, and alpha-tubulin mRNAs increased over the control levels. The results of our present study indicate that S100A4 is involved in the responses of PDL cells to mechanical stress possibly by coupling Ca2+ to the cytoskeletal system in these cells.

Biomechanical effect of crestal bone osteoplasty before implant placement: a three-dimensional finite element analysis.

This study investigated the biomechanical effects of crestal bone osteoplasty and flattening procedures carried out in edentulous knife-edge ridges to restore bone width before implant placement on the virtually placed implants using finite element methods. Three-dimensional models representing a knife-edged alveolar bone with two different crestal cortical bone thicknesses (1.6mm, thin group; 3.2mm, thick group) were created. Gradual crestal bone osteoplasty with 0.5mm height intervals was simulated. Cylindrical implants with abutments and crowns were constructed and subjected to oblique loads. Maximum stress was observed at the cervical region around the implant neck. Different osteoplasty levels showed different stress values and distributions. Highest compressive stress was observed in the flat models (60.8 MPa and 98.3 MPa in thick and thin groups, respectively), lowest values were observed when osteoplasty was limited to the sharp edge (36.8 MPa and 38.9 MPa in thick and thin groups, respectively). The results suggested that eliminating the sharp configuration in knife-edge ridges improved stress and strain outcomes, but flattening the alveolar crest and/or uncovering the cancellous bone resulted in a marked increase in compressive stress and strain values in the peri-implant bone that may influence the longevity of implants placed in these ridges.